Methods for manufacturing molecular arrays

ABSTRACT

The methods of the present invention provide methods for manufacturing a master substrate and methods for manufacturing replica arrays from the master substrate. The methods may be used, for example, directly to manufacture or “print” peptide arrays from a DNA array; however, the methods are applicable to a wide range of manufacturing applications for use any time multiple copies of an array needs to be printed.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 13/283,906, filedOct. 28, 2011.

This invention was made with the support of the Federal Government underGrant No. R44RR025296. The Federal Government has certain rights in thisinvention.

FIELD OF THE INVENTION

This invention relates to methods for manufacturing molecular arrays.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods may bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that thearticles and methods referenced herein do not constitute prior art underthe applicable statutory provisions.

Molecular arrays typically are precisely-ordered arrangements of largesets of nucleic acid, protein or other molecules immobilized on solidsubstrates, and are valuable tools in areas of research that require theidentification and/or quantification of many molecules in parallel. DNAarrays are the most common type of molecular arrays and have been usedin genetic mapping studies, mutational analyses and in genome-widemonitoring of gene expression and have become standard tools inresearch, diagnostic and clinical applications. Molecular arrays ofproteins or peptides are increasingly used in the art and areparticularly useful in high throughput screening of molecularinteractions such as protein-protein binding and enzymatic activities.

Traditionally, peptide arrays have been made by spotting pre-synthesizedpeptides on a surface (Salisbury, et al., J. Am. Chem. Soc.124(50):14868-70 (2002)) or by synthesizing peptides in spots oncellulose filter sheets using standard solid phase peptide synthesis(known as the SPOT method, see Frank, J. Immunol. Methods, 267(1):13-26(2002)). However, the cost of generating arrays with tens of thousandsor more spotted peptides would be astronomically high. Several methodsenable direct chemical synthesis of peptides in microarray format, whichreduces costs, but these methods still have the major drawback ofvariability in the quality of the synthesized peptides (Antohe andCooley, Methods Mol. Biol., 381:299-312 (2007)). Moreover, the directfabrication process can be very slow and inefficient (Hilpert, et al.,Nat. Protoc., 2:1333-49 (2007)).

Recently, methods for peptide array fabrication by in vitro translationof arrayed nucleic acids have been developed, including protein in situarray (PISA) production (He and Taussig, Nucleic Acids Res., 29: e73(2001)), nucleic acid programmable protein array (NAPPA) production(Ranachandran, et al., Science, 305:86-90 (2004)), DNA to protein array(DAPA) construction (He, Nat. Methods, 5:175-177 (2008)), and arrayingof proteins using in situ puromycin capture (Tao and Zhu, Nat. Biotech,24:1253-1254 (2006)). These approaches utilize individually-synthesizednucleic acid templates rather than individually-synthesized peptides;however, the cost of the nucleic acid templates is often higher than thecost of individual peptides arrayed by traditional methods. Further,diffusion of the peptide products limits the feature density of thesetypes of arrays.

The ability to manufacture large, high-quality, sequence-diversemolecular arrays in a cost effective manner would be of great benefitgenerally in molecular research, and in the development of diagnosticsand therapeutics in particular. The present invention addresses thisneed.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter. Other features, details,utilities, and advantages of the claimed subject matter will be apparentfrom the written Detailed Description including those aspectsillustrated in the accompanying drawings and defined in the appendedclaims.

The present invention thus provides in some embodiments a method ofmanufacturing an array of products on a substrate comprising providingpartitioned reaction volumes on the substrate, where the partitionedreaction volumes are partitioned by surface energetic barriers;generating products by one or more enzymatic processes in thepartitioned reaction volumes; and immobilizing the products from thepartitioned reaction volumes on capture moieties on the substrate, wherepartitioning between products from separate partitioned reaction volumesis preserved.

In some aspects of this embodiment there are at least 100 or morepartitioned reaction volumes per square centimeter on the substrate. Inother aspects of this embodiment, there are at least 250, 500, 750, 1000or more partitioned reaction volumes per square centimeter on thesubstrate. In yet other embodiments, there are at least 1250, 1500,1750, 2000, 2500, 3000, 4000, 5000, 7500, 10,000 or more partitionedreaction volumes per square centimeter on the substrate. In additionalembodiments, there are at least 12,500, 15,000, 17,500, 20,000, 25,000,30,000, 40,000, 50,000, 75,000, 100,000 or more partitioned reactionvolumes per square centimeter on the substrate.

In some aspects of this embodiment there are at least 100 or morefeatures per square centimeter comprising reaction products captured onthe substrate. In other aspects of this embodiment, there are at least250, 500, 750, 1000 or more features per square centimeter comprisingreaction products captured on the substrate. In yet other embodiments,there are at least 1250, 1500, 1750, 2000, 2500, 3000, 4000, 5000, 7500,10,000 or more features per square centimeter comprising reactionproducts captured on the substrate. In additional embodiments, there areat least 12,500, 15,000, 17,500, 20,000, 25,000, 30,000, 40,000, 50,000,75,000, 100,000 or more features per square centimeter comprisingreaction products captured on the substrate.

In some aspects of this embodiment, at least 25, 50, 100, 250, 500,1000, 2500, 5000, 10,000, 15,000, 20,000, 25,000 or more differentproducts from separate partitioned reaction volumes are arrayed on thesubstrate, and in other aspects at least a portion of each product fromsubstantially all partitioned reaction volumes are arrayed on thesubstrate. In additional embodiments, there are at least 30,000, 40,000,50,000, 75,000, 100,000, 150,000, 200,000 or 300,000 or more differentproducts from separate partitioned reaction volumes arrayed on thesubstrate. In yet other embodiments, there are at least 400,000,500,000, 750,000, or 1,000,000 or more different products from separatepartitioned reaction volumes arrayed on the substrate.

In some aspects of this embodiment, the surface energetic barriers areprovided on the substrate. Yet other aspects of this embodiment furtherinclude a step of introducing a second substrate in proximity to thefirst substrate where the surface energetic barriers are provided on thesecond substrate.

In some aspects of this embodiment, the one or more enzymatic processescomprise one or more of replication, transcription or translation, andin yet other embodiments, the one or more enzymatic processes compriseboth transcription and translation. In some aspects, the substratecomprises template molecules, where the template molecules comprisenucleic acids, and the products comprise nucleic acids or proteins. Insome aspects, the one or more enzymatic processes utilize one or morepolymerases.

An additional embodiment of the invention includes a method ofmanufacturing an array of products on a replica array comprisingproviding a master substrate having partitioned reaction volumes;generating products by one or more enzymatic processes in thepartitioned reaction volumes; and immobilizing the products from thepartitioned reaction volumes from the master substrate on capturemoieties disposed on the replica array that is in contact with thepartitioned reaction volumes, where partitioning between products fromseparate partitioned reaction volumes is preserved.

In some aspects of this embodiment, the reaction volumes are partitionedby surface energetic barriers on the master substrate, and in someaspects the master substrate is a template array. In yet otherembodiments, the reaction volumes are partitioned by surface energeticbarriers on a dummy substrate.

In some aspects of this embodiment, there are at least 100 or morepartitioned reaction volumes per square centimeter on the mastersubstrate. In other aspects of this embodiment, there are at least 250,500, 750, 1000 or more partitioned reaction volumes per squarecentimeter on the master substrate. In yet other embodiments, there areat least 1250, 1500, 1750, 2000, 2500, 3000, 4000, 5000, 7500, 10,000 ormore partitioned reaction volumes per square centimeter on the mastersubstrate. In additional embodiments, there are at least 12,500, 15,000,17,500, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, 100,000 or morepartitioned reaction volumes per square centimeter on the mastersubstrate.

In some aspects of this embodiment there are at least 100 or morefeatures per square centimeter comprising reaction products captured onthe replica array. In other aspects of this embodiment, there are atleast 250, 500, 750, 1000 or more features per square centimetercomprising reaction products captured on the replica array. In yet otherembodiments, there are at least 1250, 1500, 1750, 2000, 2500, 3000,4000, 5000, 7500, 10,000 or more features per square centimetercomprising reaction products captured on the replica array. Inadditional embodiments, there are at least 12,500, 15,000, 17,500,20,000, 25,000, 30,000, 40,000, 50,000, 75,000, 100,000 or more featuresper square centimeter comprising reaction products captured on thereplica array.

In some aspects of this embodiment, at least 25, 50, 100, 250, 500,1000, 2500, 5000, 10,000, 15,000, 20,000, 25,000 or more differentproducts from separate partitioned reaction volumes are arrayed on thereplica array, and in other aspects at least a portion of each productfrom substantially all partitioned reaction volumes are arrayed on thereplica array. In additional embodiments, there are at least 30,000,40,000, 50,000, 75,000, 100,000, 150,000, 200,000 or 300,000 or moredifferent products from separate partitioned reaction volumes arrayed onthe replica array. In yet other embodiments, there are at least 400,000,500,000, 750,000, or 1,000,000 or more different products from separatepartitioned reaction volumes arrayed on the replica array.

In some aspects of this embodiment, the method further includes afterthe immobilizing step, the steps of removing the replica array;sequentially furnishing one or more additional replica arrays comprisingcapture moieties; and immobilizing the products from the partitionedreaction volumes from the master substrate on the capture moieties onthe one or more additional replica arrays, where partitioning betweenproducts from separate partitioned reaction volumes is preserved.

In some aspects of this embodiment, the method is used to manufacture 2,3, 4, 5, 10, 20 or more arrays of products on replica arrays using asingle master substrate. In other embodiments, at least 30, 40, 50, 75,100, 200, 300 or more arrays of products on replica arrays aremanufactured using a single master substrate. In still otherembodiments, 400, 500, 750, 1000 or more arrays of products on replicaarrays are manufactured from a single master substrate.

In yet other aspects of this embodiment, the method further includesafter the immobilizing step, the additional steps of replenishing orregenerating partitioned reaction volumes on the master substrate;generating products by one or more enzymatic processes in thepartitioned reaction volumes on the master substrate; sequentiallyfurnishing one or more additional replica arrays comprising capturemoieties; and immobilizing the products from the partitioned reactionvolumes from the master substrate on the capture moieties on the one ormore additional replica arrays, wherein partitioning between productsfrom separate partitioned reaction volumes is preserved.

In some aspects of this embodiment, at least one component of theenzymatic reaction is attached to the master substrate. Also, in someaspects of this embodiment, the one or more enzymatic processes compriseone or more of replication, transcription or translation, and in yetother embodiments, the one or more enzymatic processes comprises bothtranscription and translation. In some aspects, the one or moreenzymatic processes utilize one or more polymerases.

Other embodiments of the invention provide a method of manufacturing anarray of products on a replica array comprising providing a templatearray comprising template molecules, a master substrate, and partitionedreaction volumes that are located between the template array and themaster substrate; generating products by one or more enzymatic processesfrom the template molecules on the template array in the partitionedreaction volumes; separating the template array from the mastersubstrate, retaining the partitioned reaction volumes on the mastersubstrate; bringing a replica array into contact with the partitionedreaction volumes; and immobilizing the products from the partitionedreaction volumes on capture moieties disposed on the replica array,wherein partitioning between products from separate partitioned reactionvolumes is preserved.

In some aspects of these embodiments, the reaction volumes arepartitioned by surface energetic barriers. In some aspects, the surfaceenergetic barriers are located on one or more of the template array, themaster substrate, the replica array or a dummy substrate.

In some aspects of this embodiment, there are at least 100 or morepartitioned reaction volumes per square centimeter on the mastersubstrate. In other aspects of this embodiment, there are at least 250,500, 750, 1000 or more partitioned reaction volumes per squarecentimeter on the master substrate. In yet other embodiments, there areat least 1250, 1500, 1750, 2000, 2500, 3000, 4000, 5000, 7500, 10,000 ormore partitioned reaction volumes per square centimeter on the mastersubstrate. In additional embodiments, there are at least 12,500, 15,000,17,500, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, 100,000 or morepartitioned reaction volumes per square centimeter on the mastersubstrate.

In some aspects of this embodiment there are at least 100 or morefeatures per square centimeter comprising reaction products captured onthe replica array. In other aspects of this embodiment, there are atleast 250, 500, 750, 1000 or more features per square centimetercomprising reaction products captured on the replica array. In yet otherembodiments, there are at least 1250, 1500, 1750, 2000, 2500, 3000,4000, 5000, 7500, 10,000 or more features per square centimetercomprising reaction products captured on the replica array. Inadditional embodiments, there are at least 12,500, 15,000, 17,500,20,000, 25,000, 30,000, 40,000, 50,000, 75,000, 100,000 or more featuresper square centimeter comprising reaction products captured on thereplica array.

In some aspects of this embodiment, at least 25, 50, 100, 250, 500,1000, 2500, 5000, 10,000, 15,000, 20,000, 25,000 or more differentproducts from separate partitioned reaction volumes are arrayed on thereplica array, and in other aspects at least a portion of each productfrom substantially all partitioned reaction volumes are arrayed on thereplica array. In additional embodiments, there are at least 30,000,40,000, 50,000, 75,000, 100,000, 150,000, 200,000 or 300,000 or moredifferent products from separate partitioned reaction volumes arrayed onthe replica array. In yet other embodiments, there are at least 400,000,500,000, 750,000, or 1,000,000 or more different products from separatepartitioned reaction volumes arrayed on the replica array.

In some aspects of this embodiment, the method further includes afterthe immobilizing step, the steps of removing the replica array;sequentially furnishing one or more additional replica arrays comprisingcapture moieties; and immobilizing the products from the partitionedreaction volumes from the master substrate on the capture moieties onthe one or more additional replica arrays, where partitioning betweenproducts from separate partitioned reaction volumes is preserved.

In some aspects of this embodiment, the method is used to manufacture 2,3, 4, 5, 10, 20 or more arrays of products on replica arrays using asingle master substrate. In other embodiments, at least 30, 40, 50, 75,100, 200, 300 or more arrays of products on replica arrays aremanufactured using a single master substrate. In still otherembodiments, 400, 500, 750, 1000 or more replica arrays of products onreplica arrays are manufactured from a single master substrate.

In yet other aspects of this embodiment, the method further includesafter the immobilizing step, the additional steps of replenishing orregenerating partitioned reaction volumes on the master substrate;generating products by one or more enzymatic processes in thepartitioned reaction volumes on the master substrate; sequentiallyfurnishing one or additional more replica arrays comprising capturemoieties; and immobilizing the products from the partitioned reactionvolumes from the master substrate on the capture moieties on the one ormore additional replica arrays, wherein partitioning between productsfrom separate partitioned reaction volumes is preserved.

In some aspects of this embodiment, the one or more enzymatic processescomprise one or more of replication, transcription or translation, andin yet other embodiments, the one or more enzymatic processes comprisesboth transcription and translation. In some aspects, the one or moreenzymatic processes utilize one or more polymerases.

Other embodiments of the invention provide a protein array comprising atleast 50,000 unique enzymatically generated protein products at adensity of at least 10,000 features per square centimeter.

In some aspects of this embodiment, at least 10,000, 20,000, 25,000,40,000 or more unique enzymatically generated protein products areprovided, and in yet other aspects, at least 75,000, 100,000, 200,000,250,000, 500,000, 1,000,000 or more unique enzymatically generatedprotein products are provided on the protein array. In some aspectsunique protein products are provided at a density of at least 100, 250,500, 750, 1000, 1250, 2000, 2500, 3000, 4000, 5000, 7500, 10,000 or morefeatures per square centimeter, and in yet other aspects, unique proteinproducts are provided at a density of at least 12,500, 15,000, 20,000,25,000, 30,000, 40,000, 50,000 75,000, 100,000 or more features persquare centimeter on the protein array.

These and other methods for manufacturing and copying molecular arraysare described in more detail herein.

DESCRIPTION OF THE FIGURES

FIGS. 1A through 1D illustrate simplified methods for manufacturingmolecular arrays according to the invention.

FIG. 2 illustrates exemplary methods for creating arrays of partitionedreaction products using a template array and one or more partitioningarrays, according to the invention.

FIGS. 3A and 3B illustrate exemplary methods for creating arrays ofpartitioned reaction volumes using various types of partitioning arrays,according to the invention.

FIGS. 4A and 4B illustrate methods of manufacturing exemplarypartitioning arrays that may be used in the methods of the invention.

FIG. 5 illustrates an exemplary method for manufacturing many replicaarrays from a single template array according to the invention.

FIG. 6 illustrates an exemplary combinatorial method for manufacturingand molecular arrays according to the invention.

FIG. 7 is a simplified illustration of another combinatorialconfiguration that may be used in methods of the invention.

FIG. 8 shows in a simplified manner yet another combinatorialconfiguration that may be employed in manufacturing molecular arrays.

It should be noted that the features of the various molecular arrays,including substrate surfaces, substrate features, droplets and the likeare not drawn to scale; rather, the features are presented in arepresentational manner.

DEFINITIONS

The terms used herein are intended to have the plain and ordinarymeaning as understood by those of ordinary skill in the art. Thefollowing definitions are intended to aid the reader in understandingthe present invention, but are not intended to vary or otherwise limitthe meaning of such terms unless specifically indicated.

The term “antibody” as used herein is intended to refer to an entireimmunoglobulin or antibody or any functional fragment of animmunoglobulin molecule that is capable of specific binding to anantigen (antibodies and antigens are “binding partners” as definedherein). Examples of such peptides include complete antibody molecules,antibody fragments, such as Fab, F(ab′)2, CDRS, VL, VH, and any otherportion of an antibody that is capable of specifically binding to anantigen. Antibodies to be used as capture moieties in the invention areimmunoreactive or immunospecific for, and therefore specifically andselectively bind to, protein products printed or copied according to theinvention.

An “array” is a group of at least two features on a substrate, and anarray may contain any number of features. The features may be molecularfeatures in the case of a molecular array, or partitioning features inthe case of a “partitioning array.” A molecular array is called a“template array” if it contains molecules (or cells, organelles, orviruses that comprise molecules) that are used as a template for abiochemical reaction such as PCR, RT-PCR, transcription, or translation.Such molecules are called “template molecules”, where such templatemolecules are, in preferred embodiments, directly or indirectly,covalently or non-covalently bound to the template array.

The term “binding pair” means any two molecules that are known toselectively bind to one another. In the case of two proteins, themolecules selectively bind to one another as described in more detailherein. Such binding may include covalent and/or non-covalent binding.Examples include, but are not limited to, biotin and avidin; biotin andstreptavidin; an antibody and its particular epitope; and the like. A“binding scaffold” refers to binding proteins generated viacombinatorial engineering and that are useful as capture moieties incertain embodiments.

The term “capture moiety” refers to any moiety that allows capture of amolecular product generated by a reaction. A substrate with capturemoieties disposed thereon is termed a capture substrate where thecapture moieties may be uniformly disposed or disposed as capturefeatures. A capture substrate is called a “replica array” if, inaddition to being capable of physically or chemically binding theproducts of a biochemical reaction involving the template molecules of atemplate array, it is an array product that is “printed” or “copied” or“stamped out” from a master substrate.

The term “copying” or “printing” or “stamping out” in reference to amolecular array means transferring a portion of molecular products froma master substrate (which may be a template array and/or partitioningarray) to a replica array.

A “feature” is an isolated region of patterned molecules or material, oran isolated depression or relief on a substrate. Features may range insize from several millimeters down to the nanometer scale depending onthe nature of the feature and the patterning technique. The areas inbetween features are referred to herein as “interstices”, or“interstitial regions.” Features may include but are not limited toisolated areas of molecules bound to or disposed upon the substrate(molecular features), and patterned regions of surface coatings such asmetal, polymer, semiconductors or insulators, regions whose surfaceenergy is markedly different from that of the interstitial region (i.e.,hydrophilic features on a hydrophobic substrate). Features may also beprovisions for separating a liquid into a plurality of compartments(i.e., separate droplets) where each compartment shall be referred to asa partition, and where a substrate comprising such compartments shall bereferred to as “partitioned.” Liquid that has been separated into dropsis described as being partitioned and a reaction in such a liquid is apartitioned reaction. A feature that is employed for the purpose ofproducing partitions in a liquid or reaction will be referred to as a“partitioning feature.” The term partitioning feature may include thefeature as well as the immediately surrounding interstitial region ifthe properties of the interstitial region contribute to forming thepartitions.

An array that is used to transform blank replica arrays into printedreplica arrays is called a “master substrate.”

The term “oligonucleotide” is used herein to mean a linear polymer ofnucleotide monomers. As used herein, the term may refer tosingle-stranded or double-stranded forms. Monomers making up nucleicacids and oligonucleotides are capable of specifically binding to anatural polynucleotide by way of a regular pattern of monomer-to-monomerinteractions, such as Watson-Crick type of base pairing, base stacking,Hoogsteen or reverse Hoogsteen types of base pairing, or the like, toform duplex or triplex forms. Such monomers and their internucleosidiclinkages may be naturally occurring or may be analogs thereof, e.g.,naturally occurring or non-naturally occurring analogs.

The terms “protein,” “peptide,” “polypeptide,” and the like are usedinterchangeably herein, and refer to a polymeric form of amino acids ofany length, comprising naturally-occurring or unnatural amino acids, orchemically or biochemically modified or derivatized amino acids,including polypeptides having modified peptide backbones.

The term “research tool” as used herein refers to any composition orassay of the invention used for scientific enquiry, academic orcommercial in nature, including the development of pharmaceutical and/orbiological therapeutics. The research tools of the invention are notintended to be therapeutic or to be subject to regulatory approval;rather, the research tools of the invention are intended to facilitateresearch and aid in such development activities, including anyactivities performed with the intention to produce information tosupport a regulatory submission.

The term “selectively binds”, “selective binding”, “specific binding”and the like as used herein, when referring to a binding partner (e.g.,protein, nucleic acid, antibody, etc.), refers to a binding reaction oftwo or more binding partners with sufficiently high affinity and/orcomplementarity to ensure selectivity or specificity under designatedassay conditions. Typically, signal that is due to specific binding willbe at least three times the standard deviation of the background signal.

The term “substrate” refers to a mechanical support upon which materialmay be disposed to provide functionality, whether mechanical,biological, optical, chemical or other functionality. A substrate may beunpatterned or patterned, partitioned or unpartitioned. Molecules on asubstrate may be disposed in features or may be uniformly disposed onthe substrate surface.

The term “surface energetic barrier” refers to a physical elementprovided by adjacent regions on a substrate having different surfaceenergetic properties that is used to partition reaction volumes.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the techniques described herein may employ, unlessotherwise indicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, sequencing technology, andmicro- and nano-fabrication which are within the skill of those whopractice in the art. Such conventional techniques include polymer arraysynthesis, hybridization and ligation of polynucleotides, and detectionof hybridization using a label. Specific illustrations of suitabletechniques can be had by reference to the examples herein. However,other equivalent conventional procedures can, of course, also be used.Such conventional techniques and descriptions can be found in standardlaboratory manuals such as Green, et al., Eds., Genome Analysis: ALaboratory Manual Series (Vols. I-IV) (1999); Weiner, Gabriel, Stephens,Eds., Genetic Variation: A Laboratory Manual (2007); Dieffenbach,Dveksler, Eds., PCR Primer: A Laboratory Manual (2003); Bowtell andSambrook, DNA Microarrays: A Molecular Cloning Manual (2003); Mount,Bioinformatics: Sequence and Genome Analysis (2004); Sambrook andRussell, Condensed Protocols from Molecular Cloning: A Laboratory Manual(2006); and Sambrook and Russell, Molecular Cloning: A Laboratory Manual(2002) (all from Cold Spring Harbor Laboratory Press); Stryer,Biochemistry (4th Ed.) (1995) W. H. Freeman, New York N.Y.; Gait,Oligonucleotide Synthesis: A Practical Approach (2002) IRL Press,London; Nelson and Cox, Lehninger, Principles of Biochemistry (2000) 3rdEd., W. H. Freeman Pub., New York, N.Y.; and Berg, et al., Biochemistry(2002) 5th Ed., W. H. Freeman Pub., New York, N.Y., Jaeger, Introductionto Microelectronic Fabrication (2002) 2nd Ed., Prentice Hall, and Madou,Fundamentals of Microfabrication (2002) all of which are hereinincorporated in their entirety by reference for all purposes.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a construct” refersto one or more copies of such construct, and reference to “the method”includes reference to equivalent steps and methods known to thoseskilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated by reference for the purpose of describing anddisclosing devices, formulations and methodologies that may be used inconnection with the presently described invention.

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in the smaller ranges, andare also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, well-known features and procedures wellknown to those skilled in the art have not been described in order toavoid obscuring the invention.

The Invention in General

The methods of the present invention provide methods for manufacturingmaster substrates and methods for printing or copying one to manyreplica arrays from a single master substrate. The methods may be useddirectly to manufacture or “print” peptide arrays from a DNA array;however, the methods are applicable to a wide range of manufacturingapplications for use any time multiple copies of an array of moleculesis desired.

The invention provides methods for spatially partitioning biochemical orchemical reactions into regions of microliter to femtoliter or smallervolumes to avoid mixing of reaction products by diffusion, convection orturbulent mixing. Partitioning is effected by the use of a partitioningarray comprising discrete partitioning features. Partitioning limitsdiffusion and preserves the spatial integrity of the reactions andresulting products. Droplets comprising a reaction mix may be assembledon the partitioning array alone, between a partitioning array and anunpartitioned substrate, or between two partitioning arrays by methodsthat are described infra. Features on the partitioning array may beseparated merely by an adequate distance to prevent droplet mixing;alternatively, in preferred embodiments the features are partitionedinto physical or virtual wells as described in more detail infra.

FIGS. 1A through 1D illustrate alternative simplified methods formanufacturing molecular arrays according to the invention. FIG. 1Aillustrates one exemplary method 100. A first step 101 provides atemplate array comprising template molecules (or cells, organelles orviruses that comprise template molecules) arrayed in features. Thetemplate array may also comprise partitioning features in which case itwould be a partitioning template array. In step 102, a substrate isprovided. The substrate may be a partitioning array comprisingpartitioning features or it may merely act as a “dummy” substrate if thetemplate array is a partitioning array. At least one of the templatearray and the substrate must be a partitioning array and both may bepartitioning arrays. At step 103, the template array and substrate arebrought into proximity with one another, and in step 104 a reaction mixis provided in the space between the template array and substrate. Thereaction mix is spatially partitioned by the partitioning features onthe template, substrate, or both. Alternatively, reaction mix may beapplied to one or both of the partitioned template array or substratebefore they are brought into proximity. At step 105 conditions areprovided so as to enable a reaction between the template moleculesdisposed on the template array and the reaction mix, thereby generatingproducts. At step 106, the template array and substrate are separatedand the fate of the products depends on the nature of the templatearray, specifically whether or not the template array contains capturemoieties. If the template contains capture moieties, the products willbecome immobilized on the surface of the template array after they aregenerated in step 105. In this case, the partitioned volumes are removedfrom the template array in step 108 and the template array has beenconverted into an array of products to be used as a research ordiagnostic tool 110. If the template array does not contain capturemoieties, the partitioned reaction products will remain in thepartitioned reaction volumes and these partitions will be preserved onthe template surface in step 109. The template thus has been convertedinto a master substrate 111, capable of being used to print replicaarrays as described in more detail infra. The substrate may be reused107 in the process with a fresh template array.

FIG. 1B illustrates an alternative exemplary method 120. In step 121, atemplate array with template molecules—or cells, organelles or virusesthat comprise template molecules—arranged in features is provided. Thetemplate array may also comprise partitioning features. In step 122, apartitioning array is provided, where the partitioning array comprisespartitioning features capable of producing partitioned reaction volumeswhere the arrangement of the partitioned volumes is compatible with thetemplate molecules in features on the template array. At step 123, thetemplate array and partitioning array are brought proximate to oneanother and in step 124 a reaction mix is provided in the space betweenthe template array and partitioning array where the reaction mix isspatially partitioned by the features on the partitioning array (or thepartitioning array and the template array if both comprise partitions).Again, as an alternative, reaction mix may be applied to one or both ofthe partitioned template array or substrate before they are brought intoproximity. At step 125 conditions are provided so as to enable areaction between the template molecules disposed on the template arrayand the reaction mix, thereby generating products. At step 126, thetemplate array and partitioning array are separated and the fate of theproducts depends on the nature of the partitioning array, specificallywhether or not the partitioning array contains capture moieties. If thepartitioning array contains capture moieties, the products have becomeimmobilized on the surface of the partitioning array after they aregenerated in step 125. In this case, the partitioned volumes are removedfrom the partitioning array in step 128 and the partitioning array thusis converted into an array of products to be used as a research ordiagnostic tool 130. If the partitioning array does not contain capturemoieties, the partitioned reaction products will remain in thepartitioned reaction volumes and these partitions will be preserved onthe partitioning array surface in step 129. The partitioning array hasbeen converted into a master substrate 121, capable of being used toprint replica arrays as described in more detail infra. The templatearray may be reused 127 in the array manufacturing method with a freshpartitioning array to generate more products.

Note that in the array manufacturing methods of the invention, eitherthe template array or the substrate or both may be partitioned; that is,the combination of partitioning template array and substrate may beemployed, the combination of (non-partitioning) template array andpartitioning array may employed, or the combination of partitioningtemplate array and partitioning array may be employed. Any configurationworks as long as the reaction mix is partitioned so that productsproduced by different template molecules reacting with the reaction mixdo not mix with one another. Note also that either the template array orthe partitioning array can be converted into a master substrate or aresearch or diagnostic tool depending on the location of thepartitioning features and the presence or absence of capture moieties.

FIG. 1C illustrates yet another alternative exemplary method 140 formanufacturing, and in this embodiment printing numerous replica arrays.In a first step 141, a master substrate, comprising products inpartitioned volumes is provided. The master substrate can be generatedaccording to the methods described supra, or by other means. In step142, a replica array is provided comprising capture moieties capable ofbinding the products from the master substrate. These capture moietiesmay be disposed in features or they may be uniformly disposed over thesurface of the replica array. The replica array may but need notcomprise partitioning features. At step 143, the master substrate andreplica array are brought proximate to one another so that the replicaarray contacts the partitioned volumes on the master substrate thatcontain the products. In step 144 a portion of the products from thepartitioned volumes is immobilized on the capture moieties of thereplica array. At step 145, the master substrate and replica array areseparated, preserving the partitioned volumes containing the products onthe master substrate. The replica array is available to be used as aresearch or diagnostic tool 146. The master substrate can then be reused147 to repeat the process at 141 as long as the partitioned volumescontain sufficient product to produce another replica array. Once themaster substrate has been depleted of products, 148, the products may bereplenished or regenerated by using one of the methods described inrelation to FIG. 1A or FIG. 1B. Note that either the template array fromFIG. 1A or the partitioning array from FIG. 1B can be used as a mastersubstrate in FIG. 1C.

The configuration of FIG. 1B is particularly useful where the templatearray comprises oligonucleotides to be transcribed and translated, and acoupled transcription/translation reaction mix is used to producepeptide products from the oligonucleotides on the template array. Thepeptide products are retained on the partitioning array, which then actsas the master substrate to print (or copy or stamp out) one to manyreplica arrays depending on how quickly the peptide products on themaster substrate (here, the partitioning array) are depleted. Using thepartitioning array rather than the template array as the mastersubstrate helps to preserve the integrity of the oligonucleotides(template molecules) on the template array so that the oligos can beused for repeated transcription/translation reactions to “load” productson additional partitioning arrays.

FIG. 1D illustrates another exemplary method 160 for manufacturingarrays where a series of reaction steps are performed. The embodimentillustrated in FIG. 1D allows for sequential addition of reactioncomponents to a partitioning array, and is particularly useful todeliver enzymes (for example, peptide tag cross-linking enzymes) orother reagents that are incompatible with other reactions that takeplace in the sequence of reactions or for reactions that requireconditions that are incompatible with other reaction conditions. In step161, a partitioning array is provided containing one or more reactioncomponents in each partitioned volume. This array can be generated byany of the methods described supra or by other methods. In step 162, afirst substrate is provided, comprising first reactants of a chemicalreaction where the reactants are also partitioned in a patterncompatible with the partitioned components on the partitioning array. Atstep 163 the partitioning array and first substrate are brought intoproximity with one another so that the partitioned volumes on thesubstrate combine with those on the partitioning array. Step 164 is anoptional step, wherein reaction conditions are provided to enable areaction between the reaction components provided by the partitioningarray and the first reactants provided by the first substrate. Thepartitioning array and substrate are separated in step 165, preservingthe partitioned reaction volumes on the partitioning array.

In step 166 a subsequent substrate is provided comprising partitionedvolumes of subsequent reactants (e.g., second substrate comprisingsecond reactants, third substrate comprising third reactants, and so on)in a pattern compatible with the partitioning array. The first substratemay be recycled 168 for this step by washing and reloading with thesubsequent reactants. In step 167 the substrate is brought intoproximity with the partitioning array as in 163 and the partitionedvolumes of subsequent reactants are combined with the partitionedvolumes on the partitioning array. Again, an optional reaction step 169,providing appropriate reaction conditions, may be included. Thesubstrate and partitioning array are separated in step 170. If thereaction requires additional components, the partitioning array isreturned to step 167 for another round of loading 171. The substrate canbe recycled 173 by washing and reloading with another round ofsubsequent reactants and reused in another cycle of 166 and 167. Oncethe final round of components has been added, final reaction conditionsare provided 169, the substrates are separated 170, and the partitioningarray is used as a master substrate or a research tool as describedsupra.

It should be noted that in the method 160 shown in FIG. 1D, conditionsmay be provided to react reaction components on the partitioningtemplate array at any point in the process; that is, conditions may beprovided after each delivery of reaction components to the partitioningtemplate array, or at one or more but not all deliveries of reactioncomponents to the partitioning template array. For example, at step 164and each time through step 169 reaction conditions may be provided.Alternatively, the reaction components on the partitioning templatearray may not be reacted before the final iteration of step 169.

Template Molecules, Reaction Mixes and Products

FIG. 2 illustrates exemplary methods for creating arrays of partitionedreaction products using template array and one or more partitioningarrays, according to the invention. In FIG. 2, at least one of thetemplate array or substrate shown comprises partitioning features andboth may comprise partitioning features.

FIG. 2 shows a template array at 202 and a substrate at 206. Templatearray 202 comprises template molecules 204. At step 201, substrate 206is brought into proximity with the template array 202 and a reaction mix208 is provided in the space between the template array 202 andsubstrate 206. At step 203, reaction mix is partitioned in the spacebetween template array 202 and substrate 206 by the partitioningfeatures provided on at least one of the template array 202 andsubstrate 206, and at step 205, template molecules 204 and reaction mix208 react to form a mixture of products and depleted reaction mix 210.In steps 207 a-207 d, the template and substrate are separated and thefate of the products in the partitioned volumes depends on the nature ofthe template array and the substrate, specifically whether eithercomprises capture moieties capable of binding the products in thepartitioned reaction volumes, and which of the template array andsubstrate retains the partitioned volumes.

Four exemplary outcomes are illustrated but others are possible. In theleftmost branch of the figure, 207 a, the template array comprisescapture moieties which capture a portion of the products 212 while thesubstrate retains the partitioned mixture 210. The template array hasbeen converted into a product array to be used as a research ordiagnostic tool and the substrate subsequently may be used as a mastersubstrate in, e.g., the methods described supra, using the rest of theproducts that were not captured by the template array. In the secondbranch, 207 b, the substrate comprises capture moieties, which capture aportion of the products 214 and the template array retains thepartitioned mixture 210. Thus, the substrate may be used as a researchor diagnostic tool and the template array may be used as a mastersubstrate. In the third branch, 207 c, neither the template array orsubstrate contains capture moieties and the template array retains thepartitioned mixture, thus the template array may be used as a mastersubstrate. Finally in the rightmost branch, 207 d, neither the templatearray nor the substrate contains capture moieties and the partitionedvolumes are retained on the substrate, thus the substrate may be used asa master substrate.

Template molecules 204 may comprise, e.g., nucleic acids, proteins,peptides, small molecules, phage, cells and the like; the reaction mix208 may comprise reaction components for PCR, transcription reactions,RT-PCR, translation reactions, or transcription reactions coupled withtranslation reactions, or enzymes, catalysts, antibodies or otherreactants; and the products in mixture 210 may comprise DNA, mRNA, cDNA,mRNA-peptide hybrids or peptides or functionalized peptides. Forexample, if the template molecule is DNA and the reaction mix comprisescomponents for PCR, the products will be DNA. In another example, if thetemplate molecule is mRNA and the reaction mix comprises components forcell-free translation, the products will be peptides. In yet anotherexample, if the template molecules are DNA and the reaction mixcomprises components for transcription coupled with translation, theproducts will be mRNA-peptide hybrids or peptides.

As should be clear given the description herein, various permutationsand combinations of template molecules, first reaction mixes, firstproducts, second and subsequent reaction mixes, second and subsequentproducts and capture moieties may be used. The products to be printedand arrayed may be nucleic acids (e.g., DNA, cDNA, oligonucleotides,RNA, mRNA, miRNAs, etc.), peptides (proteins, functionalized peptides,enzymes, antibodies, antigens, etc.) or virtually any other molecule,cell, phage or the like capable of being and desired to be configured inan array format.

Methods for producing a template array comprising template molecules arewell known in the art. For example, almost any technique for thegeneration of oligonucleotide arrays can be used, including but notlimited to, production of arrays using the Affymetrix GeneChiptechnology (Affymetrix, Santa Clara, Calif.), including techniquesdisclosed in U.S. Pat. Nos. 7,736,906, 7,691,330, 7,547,775, 5,744,305,5,677,195, 5,143,854 and U.S. Pat. Appln. Nos. 20100305006 and20090192050; Agilent microarray technologies (Agilent Technologies,Inc., Santa Clara, Calif.), including but not limited to techniquesdisclosed in U.S. Pat. Nos. 7,642,097, 7,588,889, 656,740; 6,613,893;6,599,693; 6,589,739; 6,587,579; 6,420,180; 6,387,636; 6,309,875;6,232,072; 6,221,653; and 6,180,351 and U.S. Ser. No. 20060078889;Illumina microarray technology (Illumina, Inc., San Diego, Calif.),including but not limited to synthesis techniques disclosed in U.S. Pat.Nos. 6,942,968, 6,858,394, 6,770,441, 6,429,027; and other synthesistechniques such as those disclosed in U.S. Pat. Nos. 5,807,522,5,700,637 and 5,445,934 and US Appln No. 20040259146.

The manufacturing and printing methods described herein are particularlyuseful for making peptide arrays by in vitro translation, using nucleicacid microarrays as the template array and using substrates,partitioning arrays and replica arrays to, e.g., partition thereactions, deliver reaction reagents, partition products and printreplica arrays. In standard translation reactions, purified RNA is usedas a template for translation, and the methods of the invention can beperformed using mRNAs arrayed on the template array or in sequentialmethods with DNA arrayed on the template array, and mRNA as a firstproduct. “Linked” or “coupled” systems, on the other hand, use DNA as atemplate and DNA is arrayed on the template array. In these systems, RNAis transcribed from the DNA and subsequently translated in a coupledreaction. Such systems typically combine a prokaryotic phage RNApolymerase and promoter (T7, T3, or SP6) with eukaryotic or prokaryoticextracts to synthesize proteins from exogenous DNA templates. The“linked” system may be performed as a two-step reaction, based ontranscription with a bacteriophage polymerase followed by translation inrabbit reticulocyte or wheat germ lysate. If the transcription andtranslation reactions are separate, each can be optimized to ensure thatboth are functioning at full potential. Conversely, the methods of theinvention preferably employ coupled transcription:translation reactions.

If the final products to be arrayed on replica arrays are peptides and aDNA microarray is used as a template array and are used to transcribemRNAs that are used to generate the peptide products, theoligonucleotides on the template array preferably comprise a promoterregion and a ribosome binding site (RBS) to enable translation. Optionalsequences may be included as well, such as a sequence coding for anN-terminal common peptide (for example, a TEV protease site for labelingas described in Tolbert and Wong, Agnew. Chem. Int. Ed., 41(12):2171-74(2001)) at the 5′-end of the peptide coding sequence, and/or sequencescoding for a C-terminal common peptide tag (for example, an affinity tagfor purification), as well as other sequences such as a sequenceavailable for ligation of an adaptor moiety at the 3′-end. Adaptormoieties comprising a C-terminus binding moiety such as those describedin, e.g., U.S. Pat. No. 6,416,950 to Lohse and Kurz, et al.,Chembiochem, 2:666-672 (2001), both of which are incorporated herein intheir entirety, may be used in some embodiments of the methods of theinvention.

Peptide array fabrication by in vitro translation techniques includeprotein in situ array (PISA) production (He and Taussig, Nucleic AcidsRes., 29: e73 (2001)), nucleic acid programmable protein array (NAPPA)production (Ranachandran, et al., Science, 305:86-90 (2004)), DNA toprotein array (DAPA) construction (He, Nat. Methods, 5:175-177 (2008),and arraying of proteins using in situ puromycin capture (Tao and Zhu,Nat. Biotech, 24:1253-1254 (2006)). In preferred embodiments, peptideconstructs and assay systems described in Chee and Kozlov,PCT/US2010/44134, filed Aug. 2, 2010; Chee and Kozlov, PCT/US2010/59327,filed Dec. 7, 2010; and Chee and Kozlov, U.S. Ser. No. 61/473,709, filedApr. 8, 2011 are used.

Alternatively, the template array may comprise peptide arraysmanufactured by spotting pre-synthesized peptides on the substratesurface (Salisbury, et al, J. Am. Chem. Soc. 124(50):14868-70 (2002)) orby synthesizing peptides in spots using standard solid phase peptidesynthesis (see, e.g., Frank, J. Immunol. Methods, 267(1):13-26 (2002)),or by employing other methods that enable direct chemical synthesis ofpeptides in microarray format (see, e.g., Antohe and Cooley, MethodsMol. Biol., 381:299-312 (2007)). The arrays peptides may then be used astemplates in the methods of the invention.

As described, the replica arrays of the invention comprise chemicaland/or biochemical capture moieties—typically, one element of a bindingpair—that is used to capture the products made when template moleculesreact with the reaction mix(es) of the invention. The capture moietiesof the invention include nucleic acid capture moieties such as partiallycomplementary strands for hybridization or splint ligation, DNA bindingproteins or metalloproteins, or biotin or aldehyde labeled primers, forexample.

Peptide capture moieties used to capture peptide products in someembodiments of the invention include antibody-antigen binding pairs.Alternatively, the peptide products of the invention may be modified toinclude one entity of a binding pair where the peptide capture agentcomprises the other entity of a binding pair. For example, the peptidecould comprise biotin, and the peptide capture moieties could compriseshort streptavidin binding peptides such as StrepTag (see, e.g.,Schmidt, et al., J. Mol. Bio., 255:753-66 (1996); Schmidt and Skerra, J.Chromatog. A., 676:337-345 (1994); and Skerra and Schmidt, Meth. inEnz., 326:271-304 (2000)), StrepTag II (see, e.g., Schmidt and Skerra,Nat. Protoc., 2:1528-35 (2007); and Voss and Skerra, Protein Eng.,10(8):975-82 (1997)), or HPQ motifs (see, e.g., Gissel et al., J. ofPeptide Science 1(4):217-226 (1995); and Helms et al., JBC,282(13):9813-24 (2007)). Alternatively, oligo histidine peptide tags andHis6 binding groups (see, e.g., Kneusel et al., Procedures for theAnalysis and Purification of His-tagged Proteins, in Nucleic AcidProtocols Handbook, p. 921 (2000) (Humana Press); and Smith et al.,Gene, 67:31-40 (1988)); or FLAG peptide tags and His6 or His5 peptidegroups (see, e.g., Kozlov, Combinatorial Chemistry and High ThroughputScreening, 11:24-35 (2008)); and the like may be employed with thepeptide products and peptide capture moieties of the invention.

In yet another alternative, a chemically-reactive species (e.g., analdehyde tag), label or other functionalized component may be added inthe construction of the peptide products. For example, introduction of asulfatase consensus sequence recognized by a formylglycine-generatingenzyme results in site-specific introduction of aldehyde groups into thepeptide products. The sulfatase consensus sequence can be between 6-13amino acids in length, and the smallest such “aldehyde tags” are nolarger than a His6 tag. Enzymatic modification at a sulfatase motif byformylglycine generating enzyme generates a formylglycine residue, whichallows site-specific attachment of moieties that can be captured by apeptide capture moiety. This modification is reversible, and thus theintroduction of this tag into the peptide constructs allowsaldehyde-tagged peptides to be reversibly modified with multipleepitopes. Examples of aldehyde tags for use in the present invention aredescribed in, e.g., US2008/0187956; Dierks and Frese, Chem. BioChem.,10:425-27 (2009); Wu, et al., www.pnas.orgcgi_doi_(—)10.1073_pnas.807820106; Rush and Bertozzi, J. Am. Chem. Soc.,9:130:37, (2008); Landgrebe et al., Gene, 316: 47-56 (2003); Carrico,Nat. Chem. Biology, 3:6 (2007), each of which is incorporated byreference in its entirety for teaching useful tags and their use inpeptide modification. Additionally, N-terminal formyl-methionine that isgenerated during translation initiation on all peptides can bespecifically cleaved from the peptides by peptide deformylase andmethionine aminopeptidase to expose the N-terminal cysteine. Theresulting N-terminal cysteine residue can be used for peptidemodification with an affinity residue (e.g., a biotin residue).

Also, in some embodiments, the master substrate may comprise capturemoieties, where the products from the reaction with the template arrayare captured by capture moieties on the master substrate before they areprinted or copies onto replica arrays. The printing or copying processwould encompass releasing the products from the capture moieties on themaster substrate by heat denaturing, photo-cleavage, pH change and thelike. However, in preferred embodiments, the master substrate does notcomprise capture moieties, and the products are partitioned on themaster substrate in droplets as described in detail herein.

Since many biological reactions are temperature dependent, in someembodiments controlling the temperature during the manufacture andprinting of the substrates is important. For example, in preferredembodiments, the processing temperature is kept low while the mastersubstrate is being assembled, then the processing temperature is raisedwhen reactions, such as DNA amplification, transcription, translation orother enzymatic processes, need to take place. Moreover, temperaturecontrol is one method to minimize evaporation of the template molecules,reaction mixes and products, particularly as very small volumes andfeature sizes may be employed. Other methods for minimizing evaporationare described infra.

Exemplary Reaction Partitioning Methods

A key feature of the array manufacturing methods described is an arrayof partitioned reaction volumes on a substrate created using surfaceenergetic barriers. There are many methods that can be used produce anarray of partitioned volumes. In the most simple embodiment, adrop-on-demand or ink jet method can be used to deposit templatemolecules or reaction mix(es) into the features on a partitioning array.This method can also be useful if different reaction mixes are to beused in different features. In preferred embodiments the same reactionmix is deposited in all the partitioning features, so the features canbe filled in parallel by, e.g., laminar flow, condensation of vapor orsubmerging the partitioned substrate in liquid and removing the excessliquid by wiping or spinning the partitioning array, or by evaporation.

As an alternative to drop-on-demand or ink jet printing, partitionedreactions may be formed by contact or proximity between two substrates,where one or both of the substrates are partitioning substrates.Examples for manufacturing partitioning substrates are described infra.FIG. 3A shows four alternative embodiments using contact between twosubstrates, at least one of which is a partitioning substrate. Incontact assembly, a reaction mix is added between surfaces of apartitioning array and a substrate, where the contact is sufficient toexpel excess reaction mix from the interstices. The physical barriersalone separate the reaction mix (and thus the reactions) intopartitioned reaction volumes; however, in order to be able to print manycopies of the products from a master substrate to replica arrays, it isnecessary that the partitioned reaction volumes containing the productsbe stable after one of the substrates is removed. Engineering thesurface of the partitioning array and other substrates, e.g., asdescribed in the next section by configuring slightly, moderately orvery hydrophilic or hydrophobic regions on the substrates (creatingsurface energetic barriers) is one method that may be employed.

In Scheme 1 of FIG. 3A, liquid 306 is applied to a partitioning array302, where the wells of the partitioning array 302 are moderately tovery hydrophilic. A moderately to very hydrophobic unpartitionedsubstrate 304 is then brought into contact with partitioning array 302at step 303, forcing the excess liquid out from between the partitioningarray 302 and the unpartitioned substrate 304. At step 305, thepartitioning array 302 and unpartitioned substrate 304 are separated,with the liquid 306 remaining partitioned in the hydrophilic wells ofpartitioning array 302.

In Scheme 2 of FIG. 3A, liquid 306 is applied to a partitioning array302, where the wells 310 of the partitioning array 302 are moderately tovery hydrophilic, and the interstices 308 of the partitioning array 302are moderately to very hydrophobic. A moderately to very hydrophobicunpartitioned substrate 304 is then brought into contact withpartitioning array 302 at step 303, forcing the excess liquid out frombetween the partitioning array 302 and the unpartitioned substrate 304.At step 305, the partitioning array 302 and unpartitioned substrate 304are separated, with the liquid 306 remaining partitioned in thehydrophilic wells 310 of partitioning array 302.

In Scheme 3 of FIG. 3A, liquid 306 is applied to an unpartitionedslightly hydrophilic substrate 302. A moderately to very hydrophobicpartitioning array 304 is then brought into contact with unpartitionedsubstrate 302 at step 303, forcing the excess liquid out from betweenthe unpartitioned substrate 302 and the partitioning array 304. At step305, the unpartitioned substrate 302 and partitioning array 304 areseparated, with the liquid 306 remaining partitioned on the weaklyhydrophilic surface of the unpartitioned substrate 302.

In Scheme 4 of FIG. 3A, liquid 306 is applied to a partitioning array302, having hydrophilic virtual well regions 310 and hydrophobicinterstices 308 forming surface energetic barriers between the wells. Amoderately to very hydrophobic partitioning array 404 is then broughtinto contact with partitioning array 302 at step 303, forcing the excessliquid out from between partitioning array 302 and partitioning array304. Note that the use of two partitioning arrays 302 and 304 requiresthat these arrays must be appropriately aligned. Alignment of substrates(partitioning and non-partitioning) can be achieved by methods andapparatus known in the art, such as tools similar to a contactlithography mask alignment tool and the like and as described in moredetail infra. At step 305, partitioning array 302 and partitioning array304 are separated, with the liquid 306 remaining partitioned in thehydrophilic virtual wells of partitioning array 302.

FIG. 3A thus shows four exemplary methods of contact assembly of arraysof partitioned reaction volumes using various configurations ofhydrophobic and hydrophilic regions as well as physical barriers (i.e.,various combinations of surface energetic barriers or features) to formpartitioning features. However, other characteristics such as welldepth, diameter, pitch and the like may be varied to create or enhancethe properties of the surface energetic barriers; thus, many othercombinations of these characteristics may be employed as appropriate.For example, another contact method may employ a substrate with wellsthat extend through an entire thickness of the substrate—so that thesubstrate is essentially perforated—and where flat substrates would bebrought into contact with the perforated substrate, forcing excessreaction mix out, with the remaining reaction mix disposed within theperforations. Each perforation would then represent a partitionedreaction volume.

FIG. 3B shows three exemplary embodiments for partitioning a reactionbetween two proximal substrates at least one of which is a partitioningarray. In Scheme 1 of FIG. 3B, liquid 306 is delivered to the spacebetween a partitioning array 302 and a dummy substrate 304, where thewells 310 of the partitioning array 302 are moderately to veryhydrophilic and the interstices 308 of the partitioning array 302 aremoderately to very hydrophobic. The unpartitioned dummy substrate 304 ismoderately to very hydrophobic. When dummy substrate 304 is brought intoproximity with partitioning array 302 at step 309, the reaction mix issplit into partitioned reaction volumes. This step can be aided byrapidly flowing the reaction mix into the gap between the proximalsubstrates, or by using air pressure or vacuum to displace excessreaction mixture. At step 311, the partitioning array 302 andunpartitioned substrate 304 are separated, with the liquid 306 remainingpartitioned in the hydrophilic wells 310 of partitioning array 302.

In Scheme 2 of FIG. 3B, liquid 306 is delivered between a partitioningarray 302, having moderately to very hydrophilic virtual wells and veryhydrophobic interstices 308, and a moderately to very hydrophobicunpartitioned substrate 304. The unpartitioned substrate 304 is thenbrought into proximity with partitioning array 302 at step 309, and thereaction mix is partitioned. Again, this may be aided by rapidly flowingthe reaction mix into the gap between the proximal substrates, or byusing air pressure or vacuum to displace excess reaction mixture. Atstep 311, the partitioning array 302 and unpartitioned substrate 304 areseparated, with the liquid 306 remaining partitioned in the hydrophilicvirtual wells 310 of partitioning array 302.

In Scheme 3 of FIG. 3B, liquid 306 is delivered between a partitioningarray 302, having moderately to very hydrophilic virtual wells and veryhydrophobic interstices 308, and a second partitioning array 304, alsohaving moderately to very hydrophilic virtual wells and very hydrophobicinterstices 308. The partitioning arrays 302 and 304 are then broughtinto proximity with proper alignment, and the reaction mix ispartitioned. Again, partitioning be aided by rapidly flowing thereaction mix into the gap between the proximal substrates, or by usingair pressure or vacuum to displace excess reaction mixture. At step 311,the partitioning arrays 302 and 304 are separated, with the liquid 306remaining in the hydrophilic wells 310 of both partitioned substrates302 and 304. The amount of liquid remaining on each substrated isdetermined by the geometry and surface properties of the wells on eachsubstrate. As with the embodiments for contact assembly shown in FIG.3A, the embodiments of proximity assembly shown in FIG. 3B are onlyexemplary partitioning schemes. Many other schemes, making use ofcombinations of surface properties and well depth, pitch, shape, etc.,may be employed as appropriate.

Reaction mix volumes on the substrates may vary from 10 μl to 1 fL.Reaction mix volumes and products in microliter to femtoliter volumespresent a challenge due to evaporation of the reaction mix or products.The challenge may be overcome by decreasing the temperature duringmanufacture and printing of the arrays (i.e., during non-reactiontimes); alternatively or in addition, processing may be performed in avapor-saturated environment such as a humidified glovebox.

An additional approach for stabilizing the reaction mixes and productsis to add a hydrogel-forming polymer to the reaction mixes. The hydrogelcan be cooled to solidify or gel after manufacture and during transferof substrates, but can be liquefied when the temperature is increased inorder for reactions to occur. Solidifying the partitioned reactionvolumes decreases the evaporation rate and generally protects theintegrity of the drops during processing and handling of the substrates.Generally, solidifying reaction products on a master substrate betweenreplica array printings helps to stabilize the products. One exemplaryhydrogel useful in this embodiment of the invention is an ultra-lowgelling temperature agarose such as Agarose Type IX from Sigma-AldrichCorp. (St. Louis, Mo.), which has a gelling temperature below roomtemperature and a melting temperature of approximately 50° C.,compatible with many biochemical or enzymatic reactions. In addition,use of a hydrogel provides the further advantage of enablingelectrophoretic transfer of reaction products from a master substrate toreplica arrays, thus facilitating the printing of the replica arrays.

Partitioning Arrays

A key feature of the invention is the partitioning of biochemicalreactions into arrays of discrete reaction volumes. Partitioning refersto spatially separating the biochemical reactions into partitionedreaction volumes of microliters to femtoliters to avoid mixing ofreactants or products by, e.g., diffusion, convection or turbulentmixing. In the simplest embodiment, partitioned reaction volumes areformed by depositing isolated reaction volumes at an adequate spacing toprevent merging or mixing; alternatively, in preferred embodiments thepartitioning is effected by the use of one or more partitioning arraysas described in relation to FIG. 1A through 1D and as further describedbelow. Such partitioning arrays comprise wells surrounded by barriers,where the combination of well and barrier forms a partitioning feature.Features can comprise physical wells and barriers (e.g., depressionssurrounded by ridges), or they can be virtual wells, where the featurecomprises and area of low surface energy surrounded by a high surfaceenergy barrier (e.g., a hydrophilic spot surrounded by a hydrophobicring). Partitioning features can be formed using a combination ofphysical and surface energetic elements and a partitioning array mayhave both physical and virtual features.

Partitioning arrays—like non-partitioning arrays—may consist of any of avariety of materials including glass, silicon wafers, silicone rubber,thermoplastics, and the like. The surface properties of partitioningarrays (and in some embodiments, non-partitioning arrays) are altered bychemical or physical modification and/or thin-film deposition to createthe partitioning features. In some preferred embodiments, such processesare used to create a pattern of many highly-hydrophilic regionssurrounded by highly hydrophobic regions. In the description of thepresent invention, it is assumed that reaction mixes and solvents areaqueous and thus substrate regions are described as hydrophilic orhydrophobic; however, the methods of the invention can be adapted tononpolar, fluorous or other liquids by changing the surface treatment ofthe relevant substrate.

Preferred embodiments provide partitioning arrays with patterningcreated on a micro- to nanometer scale. FIGS. 4A and 4B illustrateexemplary methods for manufacturing preferred embodiments ofpartitioning arrays. In a preferred embodiment, silicon is used for thepartitioning array due to the availability of plasma etching processesthat may be used to produce highly-hydrophilic and highly-hydrophobicnanostructured surfaces. Single crystal silicon wafers may be used ifopaque substrates are acceptable, or glass or quartz wafers withchemical vapor deposition (CVD) deposited silicon films may be used iftransparent substrates are preferred. Techniques well known to thoseskilled in the art of microfabrication are employed; for example,chemical vapor deposition, physical vapor deposition, and etch processescombined with UV-lithographic patterning permits creation ofpartitioning arrays with partitioning features as small as one micron.In other embodiments, nanostuctured patterns may also be transferred toother transparent substrates such as glass or plastic substrates bytechniques including replica-casting, hot-embossing,nanoimprint-lithography to produce transparent partitioning arrays ifrequired.

FIG. 4A shows two alternatives for manufacturing partitioning arrayswhere the partitioning features are surface energetic wells formed bypatterning of highly-hydrophilic and highly-hydrophobic materials. Asilicon wafer is shown at 402. In step 401, silicon wafer 402 issubjected to a deep reactive ion etch (DRIE) process such as a cryo-etchor Bosch-etch process to produce nanoscale pyramid or needle-likefeatures of black silicon 404 on the silicon wafer 402. At step 403, theblack silicon is chemically modified by oxidation in a furnace, oxygenplasma system or by plasma enhanced CVD (PECVD) deposition of siliconoxide to produce a superhydrophilic surface. Looking at the left branchof FIG. 4A, at step 405, the oxidized black silicon is coated with athin film of fluoropolymer 410 by, e.g., plasma deposition, to yield asuperhydrophobic surface. At step 407, a photoresist 412 is depositedand lithographically patterned to allow selective etching offluoropolymer 410. At step 409, an oxygen plasma is used to etch theexposed fluoropolymer 410 to expose the oxidized black silicon 406. Notethat the photoresist 412 is much thicker than the fluoropolymer layer soit is largely preserved despite some loss of thickness due to etching.Finally, at step 411 the photoresist 412 is removed to expose thefluoropolymer-coated oxidized black silicon 410 (i.e., superhydrophobicregions) adjacent to oxidized black silicon 406 (i.e., superhydrophilicregions). Thus, adjacent superhydrophilic and superhydrophobic regionson a micrometer scale are produced.

Looking at the right branch of FIG. 4A, in step 401, silicon wafer 402is subjected to a deep reactive ion etch (DRIE) process to producenanoscale pyramid or needle-like features of black silicon 404 on thesilicon wafer 402. At step 403, the black silicon is chemically modifiedby oxidation in a furnace, oxygen plasma system or by plasma enhancedCVD (PECVD) deposition of silicon oxide to product a superhydrophilicsurface. At step 425, a photoresist 408 is deposited in a desiredpattern to allow for lithographic patterning of the oxidized blacksilicon 406 by the process known as “liftoff” by those skilled in theart. Step 427 involves plasma deposition of a fluoropolymer 410 on theexposed areas of the oxidized black silicon 406 as well as on top of thepatterned photoresist. At step 429, the photoresist 408 is removed,exposing the regions of oxidized black silicon 406 (i.e.,superhydrophilic regions) adjacent to regions of fluoropolymer coatedblack silicon 410 (i.e., superhydrophilic regions).

FIG. 4B shows an alternative method for manufacturing a partitioningarray where the partitioning features consist of both physical and“virtual” elements. Physical depressions are etched into a siliconsubstrate that is subsequently modified to yield surface energetic wells(hydrophilic surfaces) and surface energetic barriers (hydrophobicsurfaces) aligned with the physical wells. Partitioning of substrateswith physical wells allows more control over the volume of each reactionpartition than use of virtual wells only. At step 431, a silicon water402 is patterned with photoresist 414. At step 433, areas of the siliconwafer 402 not protected with photoresist 414 are etched to produce wellsof the desired depth. At step 435, photoresist 414 is removed, and atstep 437, DRIE is performed to generate black silicon 404. At step 439,surface oxidation 406 of the black silicon is performed, and at step441, a photoresist 412 is deposited and patterned to protect the wellsof silicon wafer 402. At step 443, a fluoropolymer 410 is deposited onthe silicon wafer, and at step 445 liftoff of the photoresist 412 isperformed to expose the oxidized black silicon 406 (i.e.,superhydrophilic regions) adjacent to regions of fluoropolymer coatedblack silicon 410 (i.e., superhydrophilic regions).

It should be noted that there are many methods available in the art toproduce superhydrophobic and superhydrophilic (surface energetic)surfaces including, but not limited to direct patterning byelectron-beam lithography and plasma etching, replication bynano-imprint lithography or soft lithography, or deposition of materialby chemical-vapor deposition, sputtering or sol-gel processes.Partitioning arrays may be formed by these methods as well as others,and the exemplary methods shown are but a few.

In the methods of the invention, the size of the various substratesincluding master substrates, template arrays, partitioning arrays andcapture arrays may range from 1 mm to 1000 mm, or 1 mm to 300 mm, or 10mm to 150 mm, or 25 mm to 75 mm. In preferred embodiments each of thesubstrates (i.e. master and capture, or partitioning and template) willbe the same size and have the same number of features. In otherembodiments, one or more of the substrates may be differently sized ornot all of the features on each substrate may be used. Partitioningfeature sizes may range from 1 mm to 100 nm, or 1 mm to 1 μm, or 100 μmto 1 μm, or 100 μm to 10 μm, with partition volumes ranging from 10 μLto 1 fL, or 1 μL to 100 fL, or 1 μL to 1 pL, or 100 pL to 10 pL.Template features may be smaller, equally sized or larger than thecorresponding partitioning features as the partitioned reaction volumescan completely cover the template feature, or a portion may extendoutside the partition. Product feature size is determined by the area ofthe interface of the partitioned reaction volume and the capturemoiety-bearing surface, be it the partitioning array or a replica array.In preferred embodiments the product features are approximately the samesize as the partitioning features. Center-to-center distances betweenpartitioning features, or “pitch” can range from 10 mm to 100 nm, or 1mm to 1 μm, or 100 μm to 1 μm, or 100 μm to 10 μm. In preferredembodiments, template features, and thus product features have the samepitch as the partitioning features. Partitioning feature, templatefeature and product feature density may range from 0.01 features per mm²to 50 features per μm², 1 feature per mm² to 1 feature per μm², 10features per μm² to 0.1 features per μm², or 100 features per mm² to10,000 features per mm². Exemplary designs of product arrays produced bythe methods described include quartz, silicon or float-glass microarrayslide format substrate (25 mm×75 mm×1 mm) with 100,000 distinct proteinfeatures. The size of each feature is 50 um and the pitch of thefeatures is 100 um. A second example is a 6″ silicon wafer patternedwith 10 million protein features with a 20 um feature size and 40 umpitch.

Printing Replica Arrays

FIG. 5 illustrates an exemplary method for manufacturing many replicaarrays according to the invention. The left and right sides representtwo different exemplary methods. In FIG. 5 on the lefthand side, apartitioning array 502 with virtual wells 504 is brought into proximitywith a template array 508 with template molecules 524, with reaction mix506 delivered into the space between the partitioning array 502 andtemplate array 508. At step 501, the reaction mix is partitioned andexcess is removed from between the partitioning array 502 and templatearray 508. At step 503, the template molecules 524 disposed on templatearray 508 are allowed to react with the partitioned reaction mix 506.

In FIG. 5, on the righthand side, a partitioning array 502 with virtualwells is brought into proximity with an unpartitioned substrate 530,with reaction mix 506 delivered into the space between the partitioningarray 502 and substrate 530. Substrate 530 acts as a dummy substrateproviding a surface to allow reaction mix 506 to be partitioned onpartitioning array 502. At step 501, dummy substrate 530 is removed,leaving reaction mix partitioned on the partitioning array 502. Templatearray 508 comprising template molecules 524 is brought into proximitywith the partitioning array 502, where the template molecules 524disposed on the template array 508 are allowed to react 503 with thereaction mix 506. Note that the lefthand and righthand sides of FIG. 5use slightly different methods, yet arrive at the same point.

At step 505, the template array 508 is removed, preserving partitionedvolumes of depleted reaction mix containing products 510 on partitioningarray 502, which now may act as a master substrate for printing one ormore replica arrays. After removal, the template array 508 can bere-used as a source of template molecules to be brought into proximitywith yet another substrate (as here, a partitioning array). At step 507,a replica array 512 comprising capture moieties is brought intoproximity with the partitioning array 502 (now master substrate 502),where products in the partitioned volumes 510 on the master substrate502 can be captured by capture moieties on the replica array 512. Thepartitioning array (now master substrate 502), is used to print or stampout replica arrays. Replica array 512 is brought into proximity withmaster substrate 502, contacting partitioned volumes 510 in a mannerthat preserves the integrity of the partitioned volumes 510 whileallowing the capture of products by the capture moieties on the replicaarray 512. The proximity between replica array 512 and master substrate502 is maintained for a period of time sufficient to allow theimmobilization of a desired fraction of products from the partitionedvolumes 510 on replica array 512.

At step 509, the master substrate 502 comprising partitioned volumes 510and the replica array 512 comprising captured products 516 areseparated. At this point, the replica array 512 can be used at step 513as a research or diagnostic tool using captured products 516, and themaster substrate 502 with partitioned volumes 510 can be used at step511 to copy or print more replica array 512 comprising capture moieties,until at step 515 products are depleted (shown as depleted partitions520 on partitioning array 502). The number of replica arrays that can beprinted or copied from a single master substrate depends on the volumeof products, the size of the features, and the like, but as many as 2,5, 10, 25, 50, 100, 250, 500, 750, 850, 900 or 1000 or more replicaarrays may be printed from a single master substrate without having toreplenish the master substrate with additional reactions and products.For example, the PURExpress kit (New England Biolabs) typically yieldsup to 200 μg peptide per mL of reaction mix. For a partitioned reactionwith cylindrical partitions of 60 um diameter and 60 um height, thiscorresponds to 3.4e-11 grams of peptide per partition or 1.7e-14 molesof 16-mer peptide per partition. Also as an example, a surfacefunctionalized with closely packed IgG molecules, each capable ofcapturing 1-2 peptide molecules depending on its orientation withrespect to the surface, will have an areal density of ˜1.2e-11 moles persquare cm, capable of capturing 2.8-5.6e-16 moles of peptide per spot.Thus each partition in this example could produce enough peptide tocompletely functionalize 25-50 spots of equal area so each partitionedreaction can produce 25-50 replica arrays. The partitioning array 502(master substrate) comprising depleted products 520 can be re-used inthe process at step 517.

Looking at FIG. 5, it should be clear that various permutations andcombinations of partitioned and unpartitioned substrates (templatearrays, dummy substrates, partitioning arrays, replica arrays, etc.),comprising template molecules, reaction mixes, and capture moieties maybe used in the methods of the invention. One skilled in the art giventhe teachings of this specification regarding the partitioning,hydrophobicity and hydrophilicity of the various substrates, potentialtemplate molecules, reaction mixes, products, and the final character ofthe desired arrayed biomolecule products should be able to designseveral to many schemes to achieve a desired array.

FIG. 6 illustrates an exemplary combinatorial method for manufacturingmolecular arrays according to the invention. Combinatorial methods, inthe sense demonstrated in FIG. 6, allows for a sequential addition oftemplate molecules, reagents, reactants, catalysts, members of bindingpairs, capture moieties, and the like. First partitioning array 602 isshown comprising reaction mix 606 between first partitioning array 602and a substrate 604. As seen previously, substrate 604 simply acts as adummy substrate allowing reaction mix 606 to be properly partitionedwithin the wells of a first partitioning array 602. FIG. 6 also shows asecond partitioning array 622 and a second dummy substrate 624 with asecond reaction mix 626 disposed between them. At steps 601 and 621,dummy substrates 604 and 624 are removed, and at step 603 the firstpartitioning array 602 and second partitioning array 622 comprisingreaction mixes 606 and 626, respectively, are brought into proximity. Atstep 605, the first and second reaction mixes are allowed to merge orcombine into combined reaction mix 610, and at step 607, the secondpartitioning array 622 is removed, leaving first partitioning array 602comprising combined reaction mix 610. In some embodiments such as theone shown here, virtual wells of varying hydrophobic and hydrophiliccharacter or physical wells of varying depth may be used to ensure thatthe bulk of the combined reaction mix 610 remains on the desired (here,first) partitioning array.

FIG. 6 further shows a third partitioning array 632 and a third dummysubstrate 634 with a third reaction mix 636 disposed between them. Atstep 631, dummy substrate 634 is removed, and at step 609 the firstpartitioning array 602 comprising combined reaction mix 610 and thethird partitioning array 632 comprising third reaction mix 636 arebrought into proximity. At step 611 reaction mixes 610 and 636 areallowed to combine and react to produce combined reaction mix 612 (nowcomprising three different reaction mixes), and at step 613 firstpartitioning array 602 and third partitioning array 632 are separated,leaving combined reaction mix 612 disposed on first partitioning array602. This process can be repeated with additional substrates until thedesired reaction mixes and products are introduced and produced.

Again, it should be apparent to one skilled in the art given theteachings herein that the sequential nature of the method shown in FIG.6 lends itself to delivery of different template molecules, reactioncomponents, reagents, reactants, capture moieties, catalysts and thelike as needed to produce products to be arrayed. Any one of the first,second, third, etc., partitioning arrays in this example could betemplate arrays, capture substrates, or substrates used to deliverreaction components or simply used to aid in partitioning reactionmixes, etc. Also, it should be noted that conditions may be provided toreact reaction components on the first partitioning array at any pointin the process shown; that is, conditions may be provided after everydelivery of reaction components to the first partitioning array, or atone or more but not all deliveries of reaction components to the firstpartitioning array.

Further, in yet another embodiment, the character of partitionedreaction volumes may be engineered so that components of a droplet maycombine with another droplet, but the droplet solvent does not; such as,for example, using immiscible compounds for the droplets with favorablepartitioning coefficients. Such a process would allow, e.g., reagentdelivery or reagent extraction from a reaction mix.

FIG. 7 is a simplified illustration of another combinatorialconfiguration that may be used in methods of the invention. In Scheme 1of FIG. 7, an array of partitioned volumes 702 comprising differentreaction components 706, 708, 710 and 712 is brought into proximity atstep 701 with substrate 704 comprising partitioned volumes of a reactionmix 714, where the combined components and reaction mix react to formproducts 716, 718, 720 and 722. However, in Scheme 2 of FIG. 7, an arrayof partitioned volumes 732 comprising different reaction components 736,738, 740 and 742 is brought into proximity at step 701 with substrate734 comprising partitioned volumes of different reaction mixes 744, 746,748 and 750, where the combined different components and differentreaction mixes react to form products 716, 718, 720 and 722. Thus, themethods of the invention can be used to perform different reactionsusing a pair of partitioning array by customizing the contents of thepartitioned volumes on either or both of the arrays.

FIG. 8 illustrates yet another variation of a combinatorial approachthat can be used with the methods of the invention. FIG. 8 shows a firstpartitioning substrate 802, a second partitioning substrate 804 and athird partitioning substrate 806. The first, second and third substratesare different sizes or can be oriented differently to deliver different,specific reagents or template molecules to specific regions of asubstrate. For example, substrate 808 has four different regions: 810,812, 814 and 816. Region 810 comprises components from first substrate802 only. Region 812 comprises components from first substrate 810,second substrate 804 and third substrate 806. Region 184 comprisescomponents from first substrate 802 and second substrate 804, and region816 comprises components from first substrate 802 and third substrate806.

A reagent delivery system useful in methods of the invention may includeinstrumentation that allows the delivery of reagents to discrete regionsor features on the substrates. Reagent delivery systems useful inmethods of the invention may comprise imaging means, reagent deliveryhardware and control software. Reagent delivery can be achieved in anumber of different ways. Technologies for formulating and deliveringboth biological molecules and chemical reagents are known in the art,and uses of these instrument systems are known to one skilled in the artand easily adaptable to the methods of the invention. One example of asuitable reagent delivery system is the Labcyte™ Echo acoustic liquidhandler, which can be used to deliver nanoliter scale dropletscontaining biological molecules and reagents with high precision andreproducibility. The Labcyte™ Echo reagent delivery device may beintegrated into an overall system, using software to specify thelocations to which reagents should be delivered.

Other instruments that may be used for the deposition of templatemolecules and reagents onto biological samples include, but are notlimited to, ink jet spotting; mechanical spotting by means of pin, penor capillary; micro-contact printing; photochemical or photolithographicmethods (some of which have been described supra); and the like. Forseveral applications, it may be preferred to segment or sequestercertain areas of the substrates into one or more assay areas fordifferent reagent distribution and/or biological target determination.The assay areas may be physically separated using barriers or channels.

In one exemplary aspect, the reagent delivery system may be a flow-basedsystem. Flow-based systems for reagent delivery for use in the inventionmay include instrumentation such as one or more pumps, valves, fluidreservoirs, channels, and/or reagent storage cells. Reagent deliverysystems are configured to move fluid to contact a discrete section ofthe substrates. Movement of the reagents can be driven by a pumpdisposed, for example, downstream of the fluid reagents. The pump candrive each fluid reagent to (and past) the reaction compartment.Alternatively, reagents may be driven through the reaction compartmentby gravity. US Pub. Nos. 20070166725 and 20050239192 disclose certaingeneral-purpose fluidics tools that can be used with the methods of theinvention, allowing for the precise manipulation of gases, liquids andsolids to accomplish very complex analytical manipulations withrelatively simple hardware.

In a more specific example, one or more flow-cells can be attached to asubstrate from above. The flow-cell can include inlet and outlet tubesconnected thereto and optionally an external pump is used to deliverreagents to the flow-cell and across the substrate. If desired, the flowcells may be configured to deliver reagents only to certain portions ofthe substrate, restricting the amount and type of reagent delivered toany specific section of the substrate. In another alternative, amicrofluidic system can be integrated into the substrate or externallyattached on top of the substrate. Microfluidic passages for holding andcarrying fluid may be formed on and/or above the planar substrate by afluidics layer abutted to the substrate. Fluid reagents can be selectedand delivered according to selective opening and closing of valvesdisposed between reagent reservoirs.

Pumps generally include any device for moving fluid and/or reagentsdisposed in fluid. In some examples, the pump can be configured to movefluid and/or reagents through passages with small volumes (i.e.,microfluidic structures). The pump can operate mechanically by exertinga positive or negative pressure on fluid and/or on a structure carryingfluid, electrically by appropriate application of an electric field(s),or both, among other means. Exemplary mechanical pumps may includesyringe pumps, peristaltic pumps, rotary pumps, pressurized gas,pipettors, etc. Mechanical pumps may be micromachined, molded, etc.Exemplary electrical pumps may include electrodes and may operate byelectrophoresis, electroendoosmosis, electrocapillarity,dielectrophoresis (including traveling wave forms thereof), and/or thelike.

Valves generally include any mechanism for regulating the passage offluid through a channel. Valves can include, for example, deformablemembers that can be selectively deformed to partially or completelyclose a channel, a movable projection that can be selectively extendedinto a channel to partially or completely block a channel, anelectrocapillary structure, and/or the like.

An open gasket can be attached to the top of the substrate and reagentscan be injected into the gasket. Suitable gasket materials include, butare not limited to, neoprene, nitrile, and silicone rubber.Alternatively, a watertight reaction chamber may be formed by a gasketsandwiched between the substrate and a chemically inert, water resistantmaterial such as, but not limited to, black-anodized aluminum,thermoplastics (e.g., polystyrene, polycarbonate, etc), glass, etc.

In an optional embodiment, instrumentation employed in the methods ofthe invention comprises imaging means to determine features andorganization of the products on the replica arrays. If included, thedelivery system can comprise a microcircuit arrangement including animager, such as a CCD or IGFET-based (e.g., CMOS-based) imager and anultrasonic sprayer for reagent delivery such as described in US Pub. No.20090197326, which is incorporated herein by reference.

In yet another alternative, a delivery system may deliver reagents tospecific patterns on a substrate surface using semiconductor techniquessuch as masking and spraying. Specific areas on the substrate surfacecan be protected from exposure to reagents through use of a mask toprotect specific areas from exposure. Reagents may be introduced to thesubstrate using conventional techniques such as spraying or fluid flow.The use of masked delivery results in a patterned delivery scheme on thesubstrate surface. In an alternative embodiment, reagent deliveryinstrumentation may be based on inkjet printing technology. There are avariety of different ink-jetting mechanisms (e.g., thermal,piezoelectric). Sets of independently actuated nozzles can be used todeliver multiple reagents at the same time, and very high resolutionsare be achieved.

In many embodiments of the invention—particularly those where featuresizes and interstices are small—mechanisms to register and align veryprecisely the substrate for reagent delivery, two substrates to combinereagents, and/or the master substrate to the replica arrays for printingare thus important components of the instrumentation used with theinvention. Mechanisms such as the use of fiducial markers on slidesand/or other very accurate physical positioning systems can be adaptedto this purpose.

Instrumentation for use with the invention preferably comprises a suiteof software tailored to the array manufacturing and printing system.Optionally, oligonucleotide design software is used to design templatemolecules for the specific assay to be run, and may be integrated as apart of the system. Also optionally, algorithms and software for reagentdelivery and data analysis (i.e., sequence analysis) may be integratedinto a useful system. Integrated data analysis is particularly useful,as the type of dataset that is generated may be massive as a consequenceof scale. Algorithms and software tools that are specifically designedfor analysis of spatially-associated data, including pattern-analysissoftware and visualization tools, may enhance the value of the data.

In certain aspects, the methods employ a system that provides processesfor making and carrying out the quality control of reagents, e.g., theintegrity and sequence fidelity of oligonucleotides, reagents, and thelike. In particular, reagents are formulated according to factors suchas volatility, stability at key temperatures, and chemical compatibilityfor compatibility with the reagent delivery instrumentation and may beanalyzed by instrumentation integrated within the assay system.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention, nor are theyintended to represent or imply that the experiments below are all of orthe only experiments performed. It will be appreciated by personsskilled in the art that numerous variations and/or modifications may bemade to the invention as shown in the specific embodiments withoutdeparting from the spirit or scope of the invention as broadlydescribed. The present embodiments are, therefore, to be considered inall respects as illustrative and not restrictive.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperature, etc.) but some experimental errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, molecular weight is weight average molecularweight, temperature is in degrees centigrade, and pressure is at or nearatmospheric.

Example 1 Partitioning Array Fabrication

A partitioning array was fabricated from a standard silicon wafer.First, a patterned gasket comprising a boundary region surrounding thearea to contain partitioning features was formed on the wafer by usingUV lithography to pattern a 20 μm-thick layer of SU-8 epoxy-basednegative photoresist on the wafer. The gasket was formed such that thepartitioning array, when brought into proximity with another substrate(a dummy substrate, template array or capture substrate or replicaarray, for example), would form a flowcell. The wafer was then baked at300° C. to make the SU-8 substantially impervious to subsequentprocessing. The wafer was then cryo-etched using reactive-ion etchingwith inductively coupled plasma (RIE/ICP) at negative 140° C. using SF₆and O₂ to form black silicon. The black silicon consisted of conicalpillars, approximately 2 μm high and 100-200 nm wide.

A conformal layer of silicon dioxide approximately 50-200 nm inthickness was then deposited on the wafer by plasma-enhanced chemicalvapor deposition, causing the black silicon layer to besuperhydrophilic. The water contact angle was found to be less than 5degrees. The silicon dioxide layer also served as a spacer layer tomitigate quenching of fluorescence from fluorescent labels that may beimmobilized on the surface of the wafer. The wafer was then coated witha fluoropolymer layer, which was patterned by liftoff. Positivephotoresist was patterned by UV-lithography to produce an array ofcircular resist features surrounded by open areas. The fluoropolymer wasdeposited by plasma-assisted deposition of C₄F₈ gas in an RIE/ICP systemat room temperature. The fluoropolymer coated the black silicon pillarswith a conformal layer approximately 5-50 nm thick to yield asuperhydrophobic surface. This surface exhibited an advancing watercontact angle of >170 degrees. The circular photoresist featuresprotected the underlying black silicon and preserved thesuperhydrophilic nature of the features once this photoresist wasremoved by washing in solvents. An additional coating of photoresist wasapplied to the wafer to protect it during mechanical processing. Thewafer was cut with a diamond saw into individual chips and two holeswere drilled in each chip with a diamond drill to provide fluid portsonce the chips were assembled into flowcells with partitioning arrays.Prior to use, the photoresist protective coating was removed withsolvents and the chip was dried and baked.

Example 2 Capture Substrate Fabrication

A borofloat glass microscope slide was used to prepare the capturesubstrate. First, the slide was cleaned using solvents and a strongacid/oxidizer bath, and were then cleaned further using a UV-Ozonecleaner (Jelight, Inc., Irvine, Calif.). The slides were functionalizedusing (3-aminopropyl)trimethozysilane (APTMS) in a vapor depositionsystem at 150° C. and 3 torr for 10 minutes. Amine functionality on thecapture substrate (chip) was confirmed by labeling with LissamineRhomdamine B Sulfonyl Chloride, an amine-reactive fluorescent dye, andimaging with a microarray scanner.

Example 3 Capture Substrate Printing

The flowcell was assembled by bringing the silicon partitioning arrayinto contact with the capture substrate so that the SU-8 gasketsurrounding the region of partitioning features contacted the edges ofthe capture substrate and the black silicon surface of the partitioningarray was held at a distance equal to the thickness of the gasket. Thepartitioning array and capture substrate were then clamped together, anda tube was connected to the output port of the flowcell, where theoutput port was formed by one of the holes drilled into the partitioningarray. The tube was then connected to a vacuum source.

A small volume of buffer containing reactive molecules, in this casefluorescein isothiocyanate (FITC), was pipetted into the input portformed by the second hole drilled into the partitioning array, where theFITC was pulled through the flow cell by the vacuum. The entire volumeof the flow-cell was filled temporarily while the buffer transited theflowcell. Most of the buffer exited the flowcell through the outputport; however, droplets of FITC-containing buffer solution remainedadhered to each the hydrophilic features of the partitioning array andspanned the height of the flowcell to contact the capture array as well.The vacuum was disconnected, the ports were sealed, and the assembly wasincubated at room temperature in the dark for one hour to allow the FITCto react with the amine groups on the capture substrate.

After one hour, the vacuum line was reconnected and several aliquots ofwash buffer were pipetted into the input port, flushing the partitioningarray and capture substrate thereby washing unbound molecules from thesurface of the capture substrate. The flowcell was then disassembled(that is, the partitioning substrate and capture substrate wereseparated), and the capture substrate was rinsed and dried. Fluorescentimaging revealed that the surface of the capture substrate was patternedcorresponding to the partitions on the partitioning array, where eachfeature showed up as a fluorescent spot. There was no discernible mixingbetween spots, as evidenced by the lack of fluorescence in theinterstitial regions of the capture substrate (data not shown).

Example 4 Synthetic Oligonucleotide Array to Peptide Array ConversionUsing Partitioned Reaction

The ability to achieve peptide arrays of high density was demonstratedby converting DNA arrays using in situ coupled translation andtranscription in a partitioned reaction. Partitioning allowed forfeature densities much greater than methods previously described in theart.

DNA array preparation: Single-stranded oligonucleotide templatesencoding peptide sequences of interest were synthesized at individualfeatures on the surface of a microarray bearing surface energeticbarriers in the form of hydrophilic spots surrounded by hydrophobicinterstitial regions. Using methods known in the art, many DNA templatesmay be synthesized in parallel. Each of the templates had universaloligo sequences at the 5′ and 3′ ends. A universal sequence was used forattachment of a universal untranslated DNA region linked to a groupcapable of peptide capture. The universal oligo sequence at the 3′ endcoded for an affinity tag and a stop codon. A sequence coding for apeptide of interest was located between the universal oligo sequences atthe 5′ and 3′ ends. A third universal DNA sequence containing anuntranslated region (UTR) with a T7 promoter and a ribosomal bindingsite (RBS) was attached to each template, and a universal primercomplementary to the universal oligo sequence at the 3′ end was annealedto each template. Double-stranded DNA was formed via extension of thisuniversal primer by DNA polymerase. A capture group was introduced atthe end of one of the DNA strands on the array.

Conversion to peptide array: The DNA array was assembled in a flow-cellconfiguration to allow a small volume of reagent to be presented to thesurface in a laminar flow modality. Energetic barriers were employed onthe DNA array in addition to an extremely low-energy opposing surfacewhich forced the coupled transcription/translation (TNT) mixture to besplit into partitioned reaction volumes where the footprint of eachpartition corresponded to the area of a single DNA feature. The doublestranded oligonucleotides formed on the array surface were used astemplates for the in vitro coupled transcription/translation reaction.After the flow-cell was filled with TNT mixture (PURExpress, NEB,Ipswich, Mass.) and the mixture was partitioned, the flow-cell wasincubated at 37° C. for 30 minutes. The reaction resulted in encodedpeptides fused to an affinity tag at their C-terminus that was bound tothe capture group on the DNA array, generating of an array of peptidesattached to their own DNA templates.

By employing a partitioned reaction with only one template species perpartition, the diffusion of products between features was prevented andit was demonstrated that the density of features can be increaseddramatically above that of peptide arrays currently used in the art.Here, a template with 60 μm diameter DNA features with a spacing of 15μm between the edges of adjacent features was used. The DNA featuresthat encoded the peptides and served as templates for the peptidefeatures were surrounded by non-encoding features that were capable onlyof capturing the products via the affinity tag (capture-only features).In most cases there was no discernible level of peptide products on thecapture-only features as the products were constrained to the encodingfeatures by the energetic barriers. Thus, the size of the productfeatures was equal to the size of the DNA features, 60 μm. Using thedescribed methods, a peptide array with a density of >21,500 featuresper square centimeters can be achieved. The total number of features isonly limited by the starting oligonucleotide array. Current DNA arraytechnology allows approximately 1 million features per 2.5 cm×7.5 cmarray.

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious array manufacturing and printing arrangements which, althoughnot explicitly described or shown herein, embody the principles of theinvention and are included within its spirit and scope. Furthermore, allexamples and conditional language recited herein are principallyintended to aid the reader in understanding the principles of theinvention and the concepts contributed by the inventors to furtheringthe art, and are to be construed as being without limitation to suchspecifically recited examples and conditions. Moreover, all statementsherein reciting principles, aspects, and embodiments of the invention aswell as specific examples thereof, are intended to encompass bothstructural and functional equivalents thereof. Additionally, it isintended that such equivalents include both currently known equivalentsand equivalents developed in the future, i.e., any elements developedthat perform the same function, regardless of structure. The scope ofthe present invention, therefore, is not intended to be limited to theexemplary embodiments shown and described herein. Rather, the scope andspirit of present invention is embodied by the appended claims. In theclaims that follow, unless the term “means” is used, none of thefeatures or elements recited therein should be construed asmeans-plus-function limitations pursuant to 35 U.S.C. §112, ¶6.

1. A method of manufacturing an array of products on a replica arraycomprising: providing a master substrate having partitioned reactionvolumes; generating products by one or more enzymatic processes in thepartitioned reaction volumes; and immobilizing the products from thepartitioned reaction volumes from the master substrate on capturemoieties disposed on a replica array that is in contact with thepartitioned reaction volumes; wherein partitioning of the reactionvolumes prevents diffusion of products between them.
 2. The method ofclaim 1, wherein the reaction volumes are partitioned by surfaceenergetic barriers on the master substrate.
 3. The method of claim 1,wherein the master substrate is a template array.
 4. The method of claim1, wherein there are at least 100 partitioned reaction volumes persquare centimeter on the master substrate.
 5. The method of claim 4,wherein there are at least 10,000 partitioned reaction volumes persquare centimeter on the master substrate.
 6. The method of claim 1,wherein at least 1000 different products from separate partitionedreaction volumes on the master substrate are arrayed on the replicaarray at a density of at least 1000 different products per squarecentimeter.
 7. The method of claim 1, wherein at least a portion of eachproduct from substantially all partitioned reaction volumes are arrayedon the replica array.
 8. The method of claim 1, further comprising afterthe immobilizing step, the steps of removing the replica array;sequentially furnishing one or more replica arrays comprising capturemoieties; and immobilizing the products from the partitioned reactionvolumes from the master substrate on the capture moieties on the one ormore replica arrays, wherein partitioning between products from separatepartitioned reaction volumes is preserved.
 9. The method of claim 1,further comprising replenishing or regenerating partitioned reactionvolumes on the master substrate; generating products by one or moreenzymatic processes in the partitioned reaction volumes on the mastersubstrate; sequentially furnishing one or more replica arrays comprisingcapture moieties; and immobilizing the products from the partitionedreaction volumes from the master substrate on the capture moieties onthe one or more replica arrays, wherein partitioning between productsfrom separate partitioned reaction volumes is preserved.
 10. The methodof claim 1, wherein at least one component of at least one of theenzymatic process is attached to the master substrate.
 11. The method ofclaim 1, wherein the one or more enzymatic processes comprise one ormore of replication, transcription or translation.
 12. The method ofclaim 1, wherein the reaction volumes are partitioned by surfaceenergetic barriers on the replica array.
 13. The method of claim 1,wherein the reaction volumes are partitioned by the combination ofsurface energetic barriers on the master substrate and surface energeticbarriers on the replica array.